Abstract Development of resistance to chemotherapy was linked to persistence of cancer stem cells (CSCs). CSCs are characterized by the ability to self-renew, grow as spheres, differentiate and generate tumors in immunodeficient mice. They are resistant to traditional forms of treatment, including chemo and radio-therapy. Building on our previous work showing that ovarian CSCs residual after treatment with platinum display increased DNA methylation, we hypothesized that other epigenetic modifications occur, promote a stem-like phenotype, and render CSCs vulnerable to epigenome modifying agents. To begin to address this question, we used partial wave spectroscopy to visualize chromatin at the nanoscale level, the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to map accessible promoter regions, and proteomic analysis to measure histone marks in CSCs vs. non-CSCs. We found less open chromatin associated with repressive histone marks in CSCs vs. non-CSC and identified increased expression of the H379 histone methyl transferase Dot1L in CSCs vs. non-CSCs. Our preliminary data show that Dot1L inhibitors blocked stemness features. FOXK2, one of the transcription factors found to have an open promoter by ATAC-Seq in CSCs vs. non-CSCs was shown to be a Dot1L target gene and was highly expressed in ovarian CSCs. These preliminary observations led us to propose dissecting the link between Dot1L-induced H3K79 methylation, cancer stemness, and platinum-resistance by addressing three main objectives. Aim 1: Determine whether H3K79 methylation regulated by Dot1L is a mark and a target in OCSCs. H3K79 mono-, di-, and trimethylation will be mapped to chromatin extracted from OCSCs vs. non-CSCs by using chromatin-immunoprecipitation (ChIP)-sequencing. Effects of Dot-1L inhibitors and Dot-1L knock-down on stem cell characteristics and response to platinum will be assessed in vitro and in vivo. Aim 2: Determine whether H3K79 methylation is altered in platinum-resistant OC models and tumors. Paired parental and platinum resistant cell lines, xenografts and patient-derived xenograft models (PDX) developed by our group will be used to measure DOT1L expression and H3K79methylation. Expression and function of Dot1L will be measured in human ovarian tumors sensitive or persistent after platinum-based chemotherapy. Effects of genetic and pharmacological inhibitory strategies on response to platinum will be investigated in vitro and in vivo. Aim 3: Determine the function of FOXK2, a Dot1L target in ovarian CSCs. FOXK2 expression will be measured in platinum-resistant vs. platinum-nave models and in human ovarian tumors. Knock-in and knock- out experiments will determine its function on transcription regulation in ovarian CSCs. In summary, the proposed studies will address an important biological question with direct clinical applications. Our preliminary data and extensive expertise on cancer stem cell biology and platinum resistance strongly support the project?s feasibility and impact. We will use state of the art technologies to propel the development of new epigenome-directed strategies, such as Dot1L inhibitors, to target ovarian CSCs. Our proven track record of bench-to-clinic interventions will facilitate translation of our findings to clinical applications. The results of this project will be immediately applicable to ovarian cancer, but may have broader implications for other platinum- resistant solid tumors. In all, this project will bridge a gap of knowledge in platinum-resistant malignancies that affect a significant number of US Veterans and US military.